OligoDesign: Optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling

Abstract

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.

Figure 1

(A) Hybridization of the C.elegans cytochrome P450 LNA oligonucleotide microarray with a pool of 10 biotin-labelled antisense oligonucleotides. The 50 gene-specific CYP450 50mer LNA oligonucleotides were spotted in quadruplicate along with Cy3 labelled marker oligonucleotides (enclosed in white boxes) onto the Immobilizer microarray slide (Exiqon, Denmark). The resulting microarray was hybridized with a pool of 10 5′-biotin-labelled 50mer antisense target oligonucleotides at 65°C in 0.5 M NaCl, followed by staining with Streptavidin-Cy3 and scanning in a confocal laser scanner (ScanArray Express HT, Perkin Elmer, USA). A specific hybridization pattern is obtained from the 10 CYP450 LNA capture probes, whereas no cross-hybridization is observed for the remaining 40 CYP450 oligonucleotides. (B) Assessment of the specificity of the C.elegans CYP450 LNA oligonucleotide capture probes. Processing of the microarray data (GenePix Pro 4.01, Axon, USA) revealed strong capture probe-specific hybridization signals for the 10 CYP450 LNA oligonucleotides with a several fold higher signal intensity compared to the probe showing highest cross-hybridization.

Figure 1

(A) Hybridization of the C.elegans cytochrome P450 LNA oligonucleotide microarray with a pool of 10 biotin-labelled antisense oligonucleotides. The 50 gene-specific CYP450 50mer LNA oligonucleotides were spotted in quadruplicate along with Cy3 labelled marker oligonucleotides (enclosed in white boxes) onto the Immobilizer microarray slide (Exiqon, Denmark). The resulting microarray was hybridized with a pool of 10 5′-biotin-labelled 50mer antisense target oligonucleotides at 65°C in 0.5 M NaCl, followed by staining with Streptavidin-Cy3 and scanning in a confocal laser scanner (ScanArray Express HT, Perkin Elmer, USA). A specific hybridization pattern is obtained from the 10 CYP450 LNA capture probes, whereas no cross-hybridization is observed for the remaining 40 CYP450 oligonucleotides. (B) Assessment of the specificity of the C.elegans CYP450 LNA oligonucleotide capture probes. Processing of the microarray data (GenePix Pro 4.01, Axon, USA) revealed strong capture probe-specific hybridization signals for the 10 CYP450 LNA oligonucleotides with a several fold higher signal intensity compared to the probe showing highest cross-hybridization.